VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities

Front Immunol. 2021 Apr 1:12:657795. doi: 10.3389/fimmu.2021.657795. eCollection 2021.


Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.

Keywords: AAV (adeno-associated virus); PTM (post-translational modification); adverse drug reaction; bottom-up approach; glycosylation; immunogenicity; mass spectrometry - LC-MS/MS; proteomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Capsid Proteins / genetics*
  • Capsid Proteins / metabolism
  • Chromatography, Liquid
  • Dependovirus / genetics*
  • Dependovirus / metabolism
  • Gene Expression Regulation, Viral*
  • Genetic Engineering*
  • Genetic Vectors / genetics*
  • Genetic Vectors / isolation & purification
  • Genetic Vectors / standards
  • Glycosylation
  • Humans
  • Polysaccharides / metabolism
  • Protein Processing, Post-Translational*
  • Proteomics / methods*
  • Tandem Mass Spectrometry


  • Capsid Proteins
  • Polysaccharides